Programmable liquid, gel and biohybrid compartments and methods of use

ABSTRACT

Nano- to microscale liquid coacervate particles are provided. The liquid coacervate particles are produced by a process including stimulating a population of liquid droplets containing one or a mixture of components to induce a phase separation point of a first component, and maintaining stimulation at the phase separation point to form a coacervate domain of the first component within each of the droplets to form the liquid coacervate particles. The self-assembled nano, meso, micro and macro liquid coacervate particles and related coated substrates can have utility in drug delivery, bioanalytical systems, controlled cell culture, tissue engineering, biomanufacturing and drug discovery.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of PCT Patent Application No.PCT/US15/55836 filed Oct. 15, 2015, which claims the benefit of U.S.Provisional Application 62/064,057 filed Oct. 15, 2014, the disclosureof both of which is hereby incorporated by reference in its entirety.

FEDERAL FUNDING LEGEND

The invention was made with Government support under Federal Grant No.DMR-1121107 awarded by the National Science Foundation. The Governmenthas certain rights in the invention.

TECHNICAL FIELD

The presently disclosed subject matter relates to programmable liquid,gel and biohybrid compartments and methods of use.

BACKGROUND

Multi-phase compartments are ubiquitous within biological cells, providea powerful method for segregation of biomolecules, and are universalmotifs in synthetic polymeric particle fabrication. Unfortunately, abroad platform for the in vitro programming of complex and hierarchicalmulti-phase structures has remained elusive. Techniques such aslayer-by-layer deposition, molecular self-assembly, and microfluidicemulsion templating enable a high degree of control over the layering ofdistinct liquid or polymeric phases. However, these approaches have beenlimited to varying extents by: the need for complex and specializedfluidic devices, low fabrication throughput, limitations in achievableparticle size, and constraints on material components due to assemblyrequirements. Thus, both current microfluidic and existing bulktechniques for fabrication of hierarchical liquid-liquid, gel andparticle systems are severely lacking in scalability, size control, easeof fabrication, and morphological diversity.

SUMMARY OF THE DISCLOSURE

In some embodiments, the presently disclosed subject matter is directedto a method for making nano- to microscale liquid coacervate particles,the method comprising: stimulating a population of droplets including asolution of one or a mixture of components, wherein the stimulationinduces a phase separation point of a first component; and maintainingstimulation at the phase separation point to form a coacervate domain ofthe first component within each of the droplets, wherein liquidcoacervate particles are formed. In some embodiments, the population ofdroplets are formed using one or a combination of mechanical agitation,sonication, or microfluidics. In some embodiments, the population ofdroplets are aqueous droplets. In some embodiments, the population ofdroplets are aqueous droplets formed by sonication of the solution inoil or microfluidics of the solution in oil. In some embodiments, thepopulation of aqueous droplets are in the form of a water-in-oilemulsion. In some embodiments, the liquid coacervate particles arereversibly formed by cessation of stimulation followed by re-stimulationand re-maintaining stimulation. In some embodiments, the first componentincludes a polymer. In some embodiments, the polymer includes apolypeptide. In some embodiments, the polypeptide includes at least aportion of an elastin-like polypeptide (ELP). In some embodiments, themethod further comprises stabilizing the coacervate domain of the firstcomponent within each of the droplets to form capsule structures,wherein the coacervate domain of the first component remainsconsolidated upon cessation of stimulation at the phase separation pointof the first component. In some embodiments, stabilizing includesformation of cross-links by one or a combination of covalentcoordination, ionic interaction, disulfide bonds, or hydrogen bonds. Insome embodiments, the phase separation point is a phase separationtemperature and the stimulus includes heating. In some embodiments, theone or a mixture of components includes: a polymer, a synthetic polymer,a hydrophilic polymer, a hydrophobic polymer, an amphiphilic polymer, anamphiphilic diblock polymer, a protein, a nucleic acid, an epoxy, or apolysaccharide, and combinations thereof. In some embodiments, thestimulating includes: addition or removal of one or more of thecomponents, evaporation of the droplets, controlled diffusion of one ormore of the components, electrostatic quenching of one or more of thecomponents, inducing a reaction of one or more of the components,isomerization of one or more of the components, crosslinking of one ormore of the component, or crystallization of one or more of thecomponents, and combinations thereof. In some embodiments, a substrateis immersed within the population of aqueous droplets in the form of awater-in-oil emulsion, and a tunable degree of the coacervate domain ofthe first component is formed on a surface of the substrate based on awetting property of the substrate. In some embodiments, a graftedmolecule is present on the surface of the substrate, and the degree offormation of the coacervate domain of the first component on the surfaceof the substrate is controlled by one or both of a level of interactionof the first component with the molecule and the wetting property of thesubstrate. In some embodiments, the method further comprises stabilizingthe coacervate domain on the surface of the substrate by one or acombination of mineralization or formation of cross-links by one or acombination of covalent coordination, ionic interaction, disulfidebonds, or hydrogen bonds, wherein the coacervate domain remainsconsolidated upon cessation of stimulation at the phase separationpoint. In some embodiments, the substrate includes one or more of amedical device, a stent, a vascular graft, a catheter, a biosensor, adrug reservoir, or a cell culture substrate. In some embodiments, thepopulation of droplets are aqueous and the solution further includes oneor a combination of a cell, a virus, or a nanoparticle having a coatingof at least one component to cause recruitment of the coated cell,virus, or nanoparticle to the coacervate domain of the respectivecomponent within each of the droplets. In some embodiments, the firstcomponent has an attached bioactive agent, wherein the bioactive agentincludes one or a combination of: a drug, a protein, a peptide, apeptide hormone, a ligand, a cell-signaling ligand, or an RGD cellbinding domain, to cause recruitment of the drug, protein, peptide,peptide hormone, ligand, cell-signaling ligand, or RGD cell bindingdomain to the coacervate domain of the first component within each ofthe droplets. In some embodiments, the first component is a polypeptideand the bioactive agent is attached through an amino acid linkage orthrough a chemical linkage through a reactive peptide residue. In someembodiments, the polypeptide attached to the bioactive agent includes aprotease cleavage site. In some embodiments, the solution includes oneor more additional components each having an additional phase separationpoint, the method further comprising: stimulating the population ofaqueous droplets, wherein stimulation induces a phase separation pointof the additional component; maintaining stimulation at the additionalphase separation point to form a coacervate domain of the additionalcomponent within each of the droplets; and optionally repeating thestimulating and maintaining for one or more additional components. Insome embodiments, the first component and the additional componentsinclude polymers. In some embodiments, the polymers includepolypeptides. In some embodiments, the polypeptides include at least aportion of an elastin-like polypeptide (ELP). In some embodiments, thefirst component and the additional component(s) have similar phaseseparation points and a blended alloy coacervate domain is formed. Insome embodiments, the coacervate domain of the first component and thecoacervate domain(s) of the additional component(s) form a multilayeredcoacervate domain, a blended alloy coacervate domain, or a combinationthereof. In some embodiments, the first phase separation point and theadditional phase separation point(s) are each a phase separationtemperature, and the stimulus includes heating. In some embodiments, thesolution includes at least the first component and a surfactant forcontrolling a size of the coacervate domain. In some embodiments, thesurfactant includes an amphiphilic diblock polymer. In some embodiments,the first component is a hydrophobic ELP polymer and the amphiphilicdiblock polymer is an ELP diblock polymer. In some embodiments, a ratioof the hydrophobic ELP polymer to the amphiphilic ELP diblock polymerranges from about 1:1 to about 50:1, and the size of an outermostcoacervate domain ranges from about 50 nm to about 20 μm. In someembodiments, the method further comprises stabilizing at least anoutermost coacervate domain within each of the droplets to form capsulestructures, wherein the outermost coacervate domain remains consolidatedupon cessation of stimulation at the phase separation point for theoutermost coacervate domain. In some embodiments, stabilizing includesformation of cross-links by one or a combination of covalentcoordination, ionic interaction, disulfide bonds, or hydrogen bonds. Insome embodiments, a substrate is immersed within the population ofaqueous droplets in the form a water-in-oil emulsion, and a tunabledegree of the coacervate of the first component and the additionalcomponent(s) is formed on a surface of the substrate based on a wettingproperty of the substrate. In some embodiments, a molecule is grafted onthe surface of the substrate, and the degree of formation of thecoacervate domain of the first component and the additional component(s)on the surface of the substrate is controlled by one or both a level ofinteraction of one or both of the first component and the additionalcomponent(s) with the molecule and the wetting property of thesubstrate. In some embodiments, the coacervate domain of the firstcomponent and the additional component(s) on the surface of thesubstrate is in the form of a single layer coacervate domain, amultilayered coacervate domain, a blended alloy coacervate domain, orcombinations thereof. In some embodiments, the method further comprisesstabilizing at least an outermost coacervate domain on the surface ofthe substrate by one or a combination of mineralization or formation ofcross-links by one or a combination of covalent coordination, ionicinteraction, disulfide bonds, or hydrogen bonds, wherein the outermostcoacervate domain remains consolidated upon cessation of stimulation atthe phase separation point for the outermost coacervate domain. In someembodiments, the substrate includes one or more of a medical device, astent, a vascular graft, a catheter, a biosensor, a drug reservoir or acell culture substrate. In some embodiments, the population of dropletsare aqueous and the solution further includes one or a combination of acell, a virus, or a nanoparticle having a coating of at least onecomponent to cause recruitment of the coated cell, virus, ornanoparticle to the coacervate domain of the respective component withineach of the droplets. In some embodiments, one or more of the componentshas an attached bioactive agent, wherein the bioactive agent includesone or a combination of: a drug, a protein, a peptide, a peptidehormone, a ligand, a cell-signaling ligand, or an RGD cell bindingdomain, to cause recruitment of the drug, protein, peptide, peptidehormone, ligand, cell-signaling ligand, or RGD cell binding domain tothe coacervate domain of the component within each of the droplets. Insome embodiments, at least two components have the attached bioactiveagent. In some embodiments, the one or more components is a polypeptideand the bioactive agent is attached through an amino acid linkage orthrough a chemical linkage through a reactive peptide residue. In someembodiments, the one or more polypeptides attached to the bioactiveagent includes a protease cleavage site.

In some embodiments, the presently disclosed subject matter is directedto a method for coating a substrate, the method comprising: stimulatinga solution of one or a mixture of components, wherein a substrate isimmersed within the solution, wherein the stimulation induces a phaseseparation point of a first component; maintaining stimulation at thephase separation point to form a degree of a coacervate domain of thefirst component on a surface of the substrate based on a wettingproperty of the substrate; and repeating the stimulating and maintainingfor one or more additional components in the mixture to form acoacervate domain of the additional component. In some embodiments, thesolution is aqueous. In some embodiments, the first component and theadditional component(s) include polymers. In some embodiments, thepolymers include polypeptides. In some embodiments, the polypeptidesinclude at least a portion of an elastin-like polypeptide (ELP). In someembodiments, the first component and the additional component(s) havesimilar phase separation points and a blended alloy coacervate domain isformed on the surface of the substrate. In some embodiments, thecoacervate domain of the first component and the coacervate domain(s) ofthe additional component(s) on the surface of the substrate is in theform of a single layer coacervate domain, a multilayered coacervatedomain, a blended alloy coacervate domain, or combinations thereof. Insome embodiments, a molecule is grafted on the surface of the substrate,and the degree of formation of the coacervate domain of the firstcomponent and the additional component(s) on the surface of thesubstrate is controlled by one or both a level of interaction of one orboth of the first component and the additional component(s) with themolecule and the wetting property of the substrate. In some embodiments,the method further comprises stabilizing at least an outermostcoacervate domain on the surface of the substrate by one or acombination of mineralization or formation of cross-links by one or acombination of covalent coordination, ionic interaction, disulfidebonds, or hydrogen bonds, wherein the outermost coacervate domainremains consolidated upon cessation of stimulation at the phaseseparation point for the outermost coacervate domain. In someembodiments, the substrate includes one or more of a medical device, astent, a vascular graft, a catheter, a biosensor, a drug reservoir, or acell culture substrate. In some embodiments, the solution furtherincludes one or a combination of a cell, a virus, or a nanoparticle, andwherein the cell, virus, or nanoparticle includes a coating of at leastone of the components to cause recruitment of the cell, virus, ornanoparticle to the coacervate domain of the respective component. Insome embodiments, one or more of the components has an attachedbioactive agent, wherein the bioactive agent includes one or acombination of: a drug, a protein, a peptide, a peptide hormone, aligand, a cell-signaling ligand, or an RGD cell binding domain, to causerecruitment of the drug, protein, peptide, peptide hormone, ligand,cell-signaling ligand, or RGD cell binding domain to the coacervatedomain of the respective component. In some embodiments, at least two ofthe components have the attached bioactive agent. In some embodiments,the one or more components is a polypeptide and the bioactive agent isattached through an amino acid linkage or through a chemical linkagethrough a reactive peptide residue. In some embodiments, the one or morepolypeptides attached to the bioactive agent includes a proteasecleavage site. In some embodiments, the first phase separation point andthe additional phase separation point(s) are each a phase separationtemperature, and the stimulus includes heating. In some embodiments, theone or a mixture of components includes: a polymer, a synthetic polymer,a hydrophilic polymer, a hydrophobic polymer, an amphiphilic polymer, anamphiphilic diblock polymer, a protein, a nucleic acid, an epoxy, or apolysaccharide, and combinations thereof. In some embodiments, thestimulating includes: addition or removal of one or more of thecomponents, evaporation of the solution, controlled diffusion of one ormore of the components, electrostatic quenching of one or more of thecomponents, inducing a reaction of one or more of the components,crosslinking of one or more of the components, isomerization of one ormore of the components, or crystallization of one or more of thecomponents, and combinations thereof.

In some embodiments, the presently disclosed subject matter is directedto a coated substrate produced by a process comprising: stimulating asolution of one or a mixture of components, wherein a substrate isimmersed within the solution, wherein the stimulation induces a phaseseparation point of a first component; maintaining stimulation at thephase separation point to form a degree of a coacervate domain of thefirst component on a surface of the substrate based on a wettingproperty of the substrate; and repeating the stimulating and maintainingfor one or more additional components in the mixture to form acoacervate domain of the additional component. In some embodiments, theprocess further comprises stabilizing at least an outermost coacervatedomain on the surface of the substrate by one or a combination ofmineralization or formation of cross-links by one or a combination ofcovalent coordination, ionic interaction, disulfide bonds, or hydrogenbonds, wherein the outermost coacervate domain remains consolidated uponcessation of stimulation at the phase separation point for the outermostcoacervate domain. In some embodiments, the substrate includes one ormore of a medical device, a stent, a vascular graft, a catheter, abiosensor, a drug reservoir or a cell culture substrate.

In some embodiments, the presently disclosed subject matter is directedto a coated substrate produced by a process comprising: stimulating apopulation of aqueous droplets in the form of a water-in-oil emulsion,wherein the droplets include a solution of one or a mixture ofcomponents, wherein a substrate is immersed within the population ofaqueous droplets, and wherein the stimulation induces a phase separationpoint of a first component; maintaining stimulation at the phaseseparation point to form a tunable degree of a coacervate domain of thefirst component on a surface of the substrate based on a wettingproperty of the substrate; and repeating the stimulating and maintainingfor one or more additional components in the mixture to form acoacervate domain of the additional component. In some embodiments, theprocess further comprises stabilizing at least an outermost coacervatedomain on the surface of the substrate by one or a combination ofmineralization or formation of cross-links by one or a combination ofcovalent coordination, ionic interaction, disulfide bonds, or hydrogenbonds, wherein the outermost coacervate domain remains consolidated uponcessation of stimulation at the phase separation point for the outermostcoacervate domain. In some embodiments, the substrate includes one ormore of a medical device, a stent, a vascular graft, a catheter, a drugreservoir, a biosensor, or a cell culture substrate.

In some embodiments, the presently disclosed subject matter is directedto a nano- to microscale liquid coacervate particle composition producedby a process comprising: stimulating a population of droplets includinga solution of one or a mixture of components, wherein the stimulationinduces a phase separation point of a first component; maintainingstimulation at the phase separation point to form a coacervate domain ofthe first component within each of the droplets, wherein liquidcoacervate particles are formed; and optionally repeating thestimulating and maintaining for the one or more additional components inthe mixture to form a coacervate domain of the additional componentwithin each of the droplets. In some embodiments, the process furthercomprises stabilizing at least an outermost coacervate domain withineach of the droplets to form capsule structures, wherein the outermostcoacervate domain remains consolidated upon cessation of stimulation atthe phase separation point for the outermost coacervate domain. In someembodiments, stabilizing includes formation of cross-links by one or acombination of covalent coordination, ionic interaction, disulfidebonds, or hydrogen bonds. In some embodiments, the first component andthe additional components include polymers. In some embodiments, thepolymers include polypeptides. In some embodiments, the polypeptidesinclude at least a portion of an elastin-like polypeptide (ELP). In someembodiments, the first component and the additional component(s) havesimilar phase separation points and a blended alloy coacervate domain isformed. In some embodiments, the coacervate domain of the firstcomponent and the coacervate domain(s) of the additional component(s)form a multilayered coacervate domain, a blended alloy coacervatedomain, or a combination thereof. In some embodiments, the first phaseseparation point and the additional phase separation point(s) are each aphase separation temperature, and the stimulus includes heating. In someembodiments, the population of droplets are aqueous droplets. In someembodiments, the solution includes at least the first component and asurfactant for controlling a size of the coacervate domain. In someembodiments, the population of droplets are aqueous droplets and thesurfactant includes an amphiphilic diblock polymer. In some embodiments,the first component is a hydrophobic ELP polymer and the amphiphilicdiblock polymer is an ELP diblock polymer. In some embodiments, a ratioof the hydrophobic ELP polymer to the amphiphilic ELP diblock polymerranges from about 1:1 to about 50:1, and a size of an outermostcoacervate domain ranges from about 50 nm to about 20 μm. In someembodiments, the population of droplets are aqueous and the solutionfurther includes one or a combination of a cell, a virus, or ananoparticle having a coating of at least one component to causerecruitment of the coated cell, virus, or nanoparticle to the coacervatedomain of the respective component within each of the droplets. In someembodiments, one or more of the components has an attached bioactiveagent, wherein the bioactive agent includes one or a combination of: adrug, a protein, a peptide, a peptide hormone, a ligand, acell-signaling ligand, or an RGD cell binding domain, to causerecruitment of the drug, protein, peptide, peptide hormone, ligand,cell-signaling ligand, or RGD cell binding domain to the coacervatedomain of the respective component within each of the droplets. In someembodiments, at least two components have the attached bioactive agent.In some embodiments, the one or more components is a polypeptide and thebioactive agent is attached through an amino acid linkage or through achemical linkage through a reactive peptide residue. In someembodiments, the one or more polypeptides attached to the bioactiveagent includes a protease cleavage site.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and other features of the disclosure are explainedin the following description, taken in connection with the accompanyingdrawings, wherein:

FIG. 1A depicts droplets containing multiple fluorescently labeledcomponents according to at least one embodiment of the presentdisclosure;

FIG. 1B depicts a schematic representing mixtures of up to threecomponents in a solution according to at least one embodiment of thepresent disclosure;

FIG. 1C depicts nanoscale, mesoscale and microscale coacervate domainsevolved from droplets containing mixtures of components according to atleast one embodiment of the present disclosure;

FIG. 2 depicts time-lapse darkfield microscope images ofthermally-induced spinodal decomposition of polymer within dropletsaccording to at least one embodiment of the present disclosure;

FIGS. 3A-3O depict spinodal decomposition of multiple ELP components forformation of blended and multilayered microparticles according to atleast one embodiment of the present disclosure;

FIG. 4 depicts the formation of multi-liquid phase coacervate domainsand functionalized biomaterials according to at least one embodiment ofthe present disclosure;

FIGS. 5A-5P depicts nano-, meso- and microparticles formed by arrestedcoarsening according to at least one embodiment of the presentdisclosure;

FIG. 6 depicts isothermal and nonisothermal paths from one-phase regionto two-phase region on a phase diagram via droplet solvent evaporationaccording to at least one embodiment of the present disclosure;

FIGS. 7A-7F depicts structures formed by the droplet evaporation methodaccording to at least one embodiment of the present disclosure;

FIG. 8 depicts a schematic illustration of multilayer polymer coacervatecoatings according to at least one embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE DISCLOSURE

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to preferred embodimentsand specific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of thedisclosure is thereby intended, such alteration and furthermodifications of the disclosure as illustrated herein, beingcontemplated as would normally occur to one skilled in the art to whichthe disclosure relates.

Articles “a” and “an” are used herein to refer to one or to more thanone (i.e. at least one) of the grammatical object of the article. By wayof example, “an element” means at least one element and can include morethan one element.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this disclosure belongs.

The presently disclosed invention provides the ability to program theself-assembly of hierarchical liquid compartments comprised of polymersfrom solutions and suspensions by encoding polymer phase transitionbehavior and surface interactions at the building block level. Theability to encode information needed for programmable self-assembly intocomplex patterns is a hallmark of biological materials and emulatingthese features allows for the formation of a variety of practicallyuseful materials. The inventions of the present disclosureadvantageously affords the diversity to program phase separation eventsin liquid structures spanning the nano- to micro-scale. The presentdisclosure is valid for both synthetic and biological materials. Theinventions are demonstrated in the examples infra, which includestimulus-induced self-assembly of phase-transitioned aqueousarchitectures comprised of disordered proteins engineered viarecombinant genetics. The inventions disclosed herein enable exquisitecontrol of polymeric building blocks through the tailoring of structure,molecular weight and composition at the individual amino acid (monomer)level. The self-assembled nano, meso, micro and macro structures mayfurther find utility in drug delivery, bioanalytical systems, controlledcell culture, tissue engineering, biomanufacturing and drug discovery.

The presently disclosed invention provides the ability to program theself-assembly of hierarchical liquid coacervate particles. In oneembodiment a method is provided for making nano- to microscale liquidcoacervate particles. The method includes stimulating a population ofdroplets having a solution of one or a mixture of components to induce aphase separation point of a first component. By maintaining stimulationat the phase separation point, a coacervate domain of the firstcomponent within each of the droplets can be formed. Liquid coacervateparticles are formed through maintaining stimulation of the dropletcontaining one component or a mixture of components.

The solution can include one or more additional components each havingan additional phase separation point. The method can include furtherstimulating of the population of aqueous droplets to induce a phaseseparation point of the additional components and maintainingstimulation at the additional phase separation point to form acoacervate domain of the additional component within each of thedroplets. Optionally, repeating the stimulating and the maintaining forone or more additional components can be performed to form additionalcoacervate domains within the droplets. Examples of the liquidcoacervate particles of the invention are shown in FIGS. 1-6.

The formation of multiple all-aqueous protein-rich compartments withdistinct boundaries was directed by programming the phase behavior ofthermally-responsive elastin-like polypeptide (ELP) components (FIG. 1B)within the confinement of water microdroplets (FIG. 1A). This approachenables reversibly fabrication of (1) single- and multi-modal populationprotein compartments spanning the nano-, meso-, and micro-scale and (2)multilayered micro-architectures (FIG. 1C) by controlling phase behaviorvia simple modification of the ELP components. FIG. 1A depicts dropletscontaining multiple fluorescently labeled ELP polymers. FIG. 1B depictsa schematic representing mixtures of up to three polymers in a solution.FIG. 1C depicts nanoscale, mesoscale and microscale particles in the topportion, and multilayered microscale particles in the bottom portion,evolved from homogeneous droplets containing mixtures of ELP polymers bycontrolled phase separation and wetting.

ELPs are a class of disordered, stimuli responsive protein polymersbased on the repetitive elastin-based motif Val-Pro-Gly-Xaa-Gly (SEQ IDNO: 1), where Xaa is a tailorable guest residue. The hallmarkcharacteristic of these polymers is their lower critical solutiontemperature (LCST) phase transition behavior, where above the cloudpoint transition temperature (T sub t) they phase separate to formpolymer rich coacervate domains. For this demonstration, the strategycenters on heating through the solution coexistence boundary into theunstable region of the miscibility gap to direct phase transition viaspinodal decomposition; here, spontaneous amplification of concentrationfluctuations results in rapid formation and coarsening of protein-richdomains that are separated from a water-rich phase (FIG. 2).

FIG. 2 depicts time-lapse darkfield microscope images ofthermally-induced spinodal decomposition of polymer within water drops.Phase separated microdomains coarsen with time to form a liquid-liquidstructure comprised of a polymer-rich coacervate (dark spherical center)surrounded by a water-rich layer. Within the confinement ofmicrodroplets, spinodal decomposition provides a powerful approach forbuilding compartmentalized liquid-liquid structures through themodulation of polymeric component phase behavior. It should be notedthat other viable phase separation mechanisms include: nucleation andgrowth (metastable region); multi-component mixing-driven phaseseparation via component addition or removal, solvent evaporation,and/or controlled diffusion; electrostatic quenching; reaction-induced;crosslinking; and crystallization.

In one embodiment, a nano- to microscale liquid coacervate particlecomposition is provided that is produced by a process includingstimulating a population of droplets including a solution of one or amixture of components, wherein the stimulation induces a phaseseparation point of a first component and maintaining stimulation at thephase separation point to form a coacervate domain of the firstcomponent within each of the droplets. In this manner liquid coacervateparticles are formed. Optionally, the method includes repeating thestimulating and maintaining for the one or more additional components inthe mixture to form a coacervate domain of the additional componentwithin each of the droplets.

The population of droplets can be formed using one or a combination ofmechanical agitation, sonication, or microfluidics. The population ofdroplets can be formed by sonication of a solution in oil ormicrofluidics of a solution in oil. The population of droplets can beaqueous. The aqueous droplets can be in the form of a water-in-oilemulsion.

The liquid coacervate particles can be reversibly formed by cessation ofstimulation followed by re-stimulation and re-maintaining thestimulation. Such methods can be useful as bioreactors or fortemporarily sequestering reactants.

The programming of hierarchical aqueous polymeric layers immiscible notonly with the surrounding water solvent, but also orthogonal to otherco-existing polymeric phases is demonstrated. The results reveal thephase tunability of polymer mixtures through simple tailoring of themonomer (amino acid) sequence and molecular weight. Five differenthomopolymers (labeled U1 through U5) were used, each with distinct phasediagrams. FIGS. 3A-3F depict spinodal decomposition of multiple ELPcomponents for formation of blended and multilayered microparticles.FIGS. 3A-3C depicts temperature-controlled spectrophotometry of bulk ELPsolutions. The bulk phase transition temperatures of various homopolymermixtures are characterized using spectrophotometry (FIGS. 3A-3C). Amixture of U3 and U4 shows two independent spinodal decompositionevents, as depicted in FIG. 3A. A mixture of U1, U2, and U3 shows threeindependent spinodal decomposition events, as depicted in FIG. 3B. Amixture of U2 and U5 shows a single spinodal decomposition event, asdepicted in FIG. 3C.

When the cloud point temperatures of different components within ahomopolymer mixture are sufficiently separated from one another, observemultiple phase separation events were observed (FIGS. 3A and 3B, solidcurves) that correspond to the phase separation events of theirrespective individual components (dashed curves). Conversely,simultaneous phase separation (FIG. 3C, solid curve) of two ELPhomopolymers were observed in a mixture if the transition temperature oftheir individual components is similar (dashed curves). By carefullytraversing these multicomponent phase diagrams it was possible tocontrollably form either (1) multilayered or (2) blended ‘alloy’coacervates via discretized heating and selectively inducing variousphase separation events as predetermined in bulk.

FIGS. 3D-3O depict fluorescent microscope images of hierarchicallystructured microparticles. Double-layered coacervates made of U4(lightest coacervate in far-right image) and U3 (larger coacervateencapsulating U4 in far-right image) are depicted in FIGS. 3D-3G.Triple-layered coacervates made of U1 (light coacervate of middle-leftimage), U2 (coacervate of middle-right image encapsulating U1), and U3(coacervate of far-right image encapsulating U2) are depicted in FIGS.3H-3K. Alloy coacervates made of U2 and U5 are depicted in FIGS. 3L-3O.The inset images of FIGS. 3D-3O depict fluorescent channel(s) of ELPsthat have and/or are phase separating. The double-layer (FIGS. 3D-3G)and triple-layer (FIGS. 3H-3K) protein coacervates were assembled byexploiting the effects of micro-confinement on the phase separationprocess. By encapsulating mixtures of homopolymers inside droplets andsubsequently heating in a step-wise fashion, the unstable region of thephase diagram for each successive increasingly hydrophilic proteincomponent was targeted; the system is held above each transitiontemperature until phase separation of the target protein proceeds tocompletion. Subsequent phase separation events in this manner result inthe formation of n coacervate layers, where n is the number of polymericcomponents inside the droplets with dissimilar phase diagrams. Only onepolymer type populates each phase-transitioned layer.

The formation of wetting architectures is described by componentinterfacial tensions (γ sub i/j) and the spreading parameter (S):

S=γsub(IP/Water)−(γsub(IP/MP)+γsub(MP/Water)).

A positive spreading parameter (S>0) results in the formation of wettinglayers, which is due to a high degree (≧80% by composition) of chemicalhomology between the protein polymers. By contrast, a negative spreadingparameter (S<0) will result in Janus structures. Thus, interfacialenergies and the accompanying spreading behavior are a key module forstructural design with the method presented herein.

In contrast to distinct layered liquid phases, protein coacervate‘alloy’ blends were formed when the phase transition is initiated for abinary mixture comprised of two different polymers with differentchemical composition but with similar phase transition temperatures, asevidenced by inspection of FIGS. 3L-3O. Thus, both physical and chemicalproperties can be engineered into each liquid phase layer by tailoringthe system phase behavior at the building block level.

The types and spatiotemporal distributions of signaling moleculesdetected by target cells in extracellular regulation are the subject ofextensive investigations. Thus, there is a growing need for creatingdiverse extracellular matrices (ECMs) from an array of programmablebiologically functional building blocks, with versatile modular design.The method of manipulating polymer assembly can allow (i)high-throughput encapsulation of cells, cellular components (e.g.,organelles) or viruses within biocompatible (e.g., polypeptide) gelmicroarrays, (ii) capturing cells, cellular components (e.g.,organelles) or viruses within tailorable layers of single population or‘alloy’ blends of gels, each with distinct intrinsic physicochemicalfunctionalization, (iii) capturing cells, cellular components (e.g.,organelles) or viruses within capsule-like structures with modular coreand shell properties, (iv) spatial organization of immobilizedcell-signaling ligands and within the gel matrix, (v) tailoring of gelECM mechanical properties, (vi) integration of degradable (e.g.,protease-cleavable) linkages, and (vii) combinations thereof (shownschematically in FIG. 4). In FIG. 4, the dotted triangle representsintegrin ligands (recreate cell-cell and cell-ECM interactions), theblack triangle represents immobilized soluble biomolecules (growthfactors, cytokines), the white triangle represents recapitulatedcell-cell interactions (ephrins, cadherins, CAMs), and X representsprotease sensitive degradation sites. The flexibility of these methodscan be taken advantage of for forming multi-liquid phase compartments tocreate functionalized biomaterials for ECM assembly that have potentialfor direct translation to regenerative medicine, cell-culture,cell-based therapies, drug discovery, drug delivery, and tissueengineering related markets.

The specific characteristics of phase transitioned liquid-liquid phasesdepend strongly on molecular and surface interactions. Validating thiswas possible by utilizing protein diblock surfactants (called D1) toself-assemble populations of uniform polymer-rich compartments withcontrollable size. FIGS. 5A-5P depict nano-, meso- and microparticlesformed by arrested coarsening. FIGS. 5M-5N depicts a schematic showingsequences of the ELP diblock (FIG. 5M) and U1 uniblock (FIG. 5N). TheELP diblocks consist of two adjoined blocks of dissimilar amino acidrepeats. The amphiphilic nature of these diblock polypeptides enablesthe temperature-triggered self-assembly of nanoscale micelles (D≈56 nm)when D1 is the lone component. FIGS. 5A-5D depicts fluorescentmicroscope images of water drops containing various ratios ofdarker-labeled ELP diblock and lighter-labeled U1. The diblock arrestscoarsening of phase-separated U1 uniblock domains by acting as asurface-stabilizing agent. In the case of a homopolymer/diblock mixture,phase-separated polypeptide compartments that span the nano-, meso-, andmicro-scale (FIGS. 5M-5N) were fabricated by simply modulating thestoichiometric ratio of the components. Upon heating the mixture intothe unstable region of the homopolymer phase diagram, the homopolymerbegins to phase separate via spinodal decomposition. Coarsening of thepolymeric-rich liquid domains is arrested at a specific size by thestabilizing protein amphiphiles; the protein compartment size is afunction of diblock quantity. This strategy provides access to thegenerally less-accessible meso-scale and offers a mechanism for thefabrication of particles spanning multiple length scales via programmedphase separation.

The resultant protein compartments have a narrow size distribution(FIGS. 5I-5L), presumably due to mass conservation and molecularpacking. FIGS. 5I-5L depict a schematic of the polypeptide compartmentsformed by varying U1/diblock ratios. The diameters of the resultantprotein phase puncta are: (U1 to diblock ratio in parentheses): 56±3 nm(0:1), 625±34 nm (1:1), 3±0.6 μm (5:1), and 17±1 μm (50:1). Furthermore,these phase-transitioned domains do not experience appreciableprecipitation and are kinetically stable for up to 24 hours.

Biomaterial can be fabricated by covalently crosslinking thecoarsen-arrested protein microcompartments. Multi-modal populations ofprotein-rich water compartments can be fabricated by supplementing theaqueous precursor solution with a second population of U2 homopolymer.

By simply changing the stoichiometric ratios of mixtures U1, U2, and D1two distinct, orthogonally phase-separated populations of similar-sizedmicrocompartments were created (FIG. 5O) and a single coacervatecompartment coexisting with numerous smaller compartments (FIG. 5P);this type of multi-component protein compartmentalization can be usefulfor recreating complex non-membrane bound intracellularmicroenvironments through controlled segregation of biomacromolecules.FIGS. 5O-5P depict fluorescent microscope images of distinct uniblockpolypeptide domains. FIG. 5O consists of a bimodal population of U1(top-left inset) and U2 (top-middle inset) microcompartments with ratioof total uniblock to diblock (top-right inset) of 5:1. FIG. 5P depictsfully coarsened U1 coacervate (top-left inset) surrounded by U2microcompartments (top-middle inset) with ratio of total uniblock todiblock (top-right inset) of 50:1. Top panels of FIGS. 5O and 5P show U1(left), U2 (middle) and D1 (right) fluorescent channels; bottom panelsof FIGS. 5O and 5P are overlay images.

This technology can enable one-pot fabrication of complex hierarchicalbiological and synthetic particle architectures through preprogrammedphase separation and self-assembly. The ability to evolve complexstructures is in contrast to microfluidic or layer-by-layer methods,where each layer or phase is added in sequential steps; thus, massivescale up in production, size control, and reduced fabrication costs isfacilitated by the technology. Additionally, phase separated low-surfaceenergy water compartments, crowded with biomolecules, mimicsnon-membrane bound organelles within the cytoplasm and nucleoplasm ofcells. Thus, the system enables an avenue for a more thoroughunderstanding of the mechanisms of microdomain formation for the broadimplementation of: tissue engineering pathways through cellencapsulation, protein microdomains as intra- and extracellularswitches, protocells as bioreactors, drug delivery vehicles and depots,and fundamental studies of biological macromolecule assembly intocondensed phases. Within this paradigm, other materials include:hydrophilic and hydrophobic liquid monomers; synthetic polymers; proteinand nucleic acid components; water/polymer and water/monomer mixtures;epoxies; non-polymeric liquids; cells; organelles; viruses;polysaccharides; polymer/monomer combinations; and mixtures thereof.

FIG. 6 depicts isothermal (top arrow) and nonisothermal (bottom arrow)paths from one-phase region to two-phase region on a phase diagram viadroplet solvent evaporation.

In another embodiment, methods are provided for coating a substrate. Thesubstrate can include one or more of a medical device, a stent, avascular graft, a catheter, a biosensor, a drug reservoir, or a cellculture substrate. Examples of coating of substrates are shown in FIGS.7-8.

FIGS. 7A-7F depicts structures formed by the droplet evaporation method,including non-wetting (FIG. 7A), partially wetting (FIG. 7B) and fullywetting (FIG. 7C) phase-transitioned coacervates on a substrate surface.Additional structures can include multilayer compartments (FIG. 7D), andmicelles or other particles (FIG. 7E) and cells (FIG. 7F) encapsulatedwithin the resultant coacervates.

The approach is not limited to creating hierarchical liquid compartmentswithin emulsions using temperature-induced phase separation. Theplatform is amenable to evolving the phase-separated structurespresented herein within surface-bound drops via controlled evaporation.The strategy is to control evaporation of solvent (e.g., water) from amulticomponent droplet to move across the phase diagram from theone-phase region to the two phase region, as shown schematically in FIG.6. This methodology obviates the requirement of an external continuousfluid (e.g., oil) and provides modulated control of the components viaextent of solvent evaporation. The top arrow in FIG. 6 represents theideal horizontal isothermal path across the phase diagram into thetwo-phase region. Droplet cooling may occur due to evaporation; thebottom arrow in FIG. 6 represents a pathway whereby phase separationoccurs via a non-isothermal evaporative process. In both examples,coacervate formation is expected to occur because the final state of thesystem falls within the two-phase region of the phase diagram.

The surface chemistry and topography of the substrate is key indetermining the properties of the resultant phase-transitionedcoacervates. For example, if the coacervate does not wet the surfacepreferentially to water, a spherical coacervate to form within the dropis expected, as illustrated in FIG. 7A. Surface coatings that can resultin this non-wetting behavior include, but are not limited to:PEG-silane, PEG-thiols on gold, POEGMA brushes, and zwitterionicsurfaces. In the case of a coacervate contact angle between 0° and 180°with respect to water, the coacervate can achieve formation of a partialsphere (FIG. 7B). If the coacervate completely wets the surface, auniform film may form at the bottom of the drop upon coacervation (FIG.7C); this film could be attained by tethering polymers or oligomerss tothe surface expressing the same (or similar) physical properties tothose in the coacervate. The approach also applies to hierarchicalstructures evolved within the drops. This fabrication strategy caneasily be extended to multilayer compartments (FIG. 7D), coacervatesencapsulating micelles and other nano- to micro-particles (FIG. 7E), andcell, organelle or virus encapsulation (FIG. 7F) within the formedcoacervates.

Other examples whereby the concepts, methods and materials describedsupra can be used include the formation of multilayer, blend or micro-and nanostructured thin film coatings on surfaces of solid materials bytailoring the phase behavior and surface interactions of polymers. Ofparticular interest is the formation of such complex coatings frompeptide polymers and other bioactive polymers, which may be tailored tohave a number of desirable biological properties for biomedicalapplications.

Formation of liquid coacervate phases in the form of thin films(coatings) on a desired surface can be accomplished using any of themeans described above (temperature change, evaporation, change insolvent quality (e.g. by addition of cosolutes) by placing the surface(e.g. biomedical device) of interest to be coated into a homogeneoussolution of the polymer mixtures either before or while phase separation(coacervation) is occurring. FIG. 8 depicts a schematic illustration ofmultilayer polymer coacervate coatings. In this example, tailoring nconstituent phase transitions in multicomponent systems enablesformation of n immiscible wetting layers (n=3 above). In other examples,each layer can consist of a single component, immiscible with othercomponents or can consist of blends, where multiple chemicallyheterogeneous components are tuned to be miscible in the same layer, andcombinations thereof.

Morphology of the coating on the surface depends on the sequence ofselective coacervation (phase separation) of polymeric components insolution onto the surface of interest, and the wetting behavior of thephase separating polymer on that surface. In analogy to the structuresdepicted in FIGS. 7A-7C, wetting can determine the lateral morphology ofthe phase separating layers, so that structures can range from uniformlyspread multilayers to poorly spread layers that display a complextopography.

Examples of the types of biomedical devices that can be coated includestents, vascular grafts, catheters, biosensors, cell culture substrataor any material or device for which a biofunctional coating comprisingthe polymer layer is desired.

Examples of the types of bioactive function for these coatings include,drug delivery, drug elution, cell encapsulation, biosensing,diagnostics, theranostics, permselective membranes, lubrication layers,and templates for biomineralization of other forms of materialsself-assembly.

After (or during) formation of the polymer layer by selectivecoacervation and controlled wetting, it may be necessary to stabilizethem by crosslinking, mineralization or otherwise encapsulating them.Crosslinking may be by covalent, coordination, ionic, disulfide andhydrogen bonding for example.

One method for coating a substrate includes stimulating a solutionhaving one or a mixture of components, wherein the substrate is immersedwithin the solution, and the stimulation induces a phase separationpoint of a first component. The stimulation is maintained at the phaseseparation point to form a degree of a coacervate domain of the firstcomponent on a surface of the substrate. The degree of coacervate domainformed on the surface is based on the wetting property of the substrate.The stimulating and maintaining is repeated for one or more additionalcomponents in the mixture to form a coacervate domain of the additionalcomponent(s).

Another method for coating a substrate includes stimulating a populationof aqueous droplets in the form of a water-in-oil emulsion where thedroplets include a solution of one or a mixture of components. Thesubstrate is immersed within the population of aqueous droplets.Stimulation of the droplets induces a phase separation point of a firstcomponent and maintaining stimulation at the phase separation pointforms a tunable degree of a coacervate domain of the first component ona surface of the substrate. The tunable degree of the coacervate domainthat is formed on the surface is based on the wetting property of thesubstrate. The stimulating and maintaining is repeated for one or moreadditional components in the mixture to form a coacervate domain of theadditional component(s).

The coated substrates, formed by the methods described herein, caninclude a grafted molecule present on the surface of the substrate. Inthis case, the degree of formation of the coacervate domain of the firstcomponent, and/or additional components, on the surface of the substratecan be controlled by one or both of the level of interaction of therespective component with the molecule and with the wetting property ofthe substrate.

The coacervate domain of the first component and the additionalcomponent(s) on the surface of the substrate can be in the form of asingle layer coacervate domain, a multilayered coacervate domain, ablended alloy coacervate domain, or combinations thereof.

In one embodiment, a coated substrate is provided that is produced by aprocess including stimulating a solution of one or a mixture ofcomponents, wherein a substrate is immersed within the solution, whereinthe stimulation induces a phase separation point of a first component;maintaining stimulation at the phase separation point to form a degreeof a coacervate domain of the first component on a surface of thesubstrate based on a wetting property of the substrate; and repeatingthe stimulating and maintaining for one or more additional components inthe mixture to form a coacervate domain of the additional component.

In one embodiment, a coated substrate is provided that is produced by aprocess including stimulating a population of aqueous droplets in theform of a water-in-oil emulsion, wherein the droplets include a solutionof one or a mixture of components, wherein a substrate is immersedwithin the population of aqueous droplets, and wherein the stimulationinduces a phase separation point of a first component; maintainingstimulation at the phase separation point to form a tunable degree of acoacervate domain of the first component on a surface of the substratebased on a wetting property of the substrate; and repeating thestimulating and maintaining for one or more additional components in themixture to form a coacervate domain of the additional component.

With respect to the methods and processes described herein for formationof nano- to microscale liquid coacervate particles and coatedsubstrates, the methods and processes of formation can further includestabilizing at least an outermost coacervate domain of the droplet or ofthe outermost coacervate domain on the surface of the substrate.Stabilization can be effected by one or a combination of mineralizationor formation of cross-links by one or a combination of covalentcoordination, ionic interaction, disulfide bonds, or hydrogen bonds.After stabilization of the outermoset coacervate domain of the liquiddroplet, this domain remains consolidated upon cessation of stimulationat the phase separation point for this coacervate domain, and capsulestructures are formed. After stabilization of the outermoset coacervatedomain on the surface of the substrate, this domain remains consolidatedupon cessation of stimulation at the phase separation point for thiscoacervate domain. In addition to the outermost coacervate domains, oneor more of the additional coacervate domains can be stabilized in thedroplets and on the substrate surfaces. After stabilization, thecoacervate domain(s) can remain consolidated upon cessation ofstimulation at the respective phase separation point of the respectivecomponent(s) of the domain.

The components of the present methods and processes can include, forexample, but are not limited to, a polymer, a synthetic polymer, ahydrophilic polymer, a hydrophobic polymer, an amphiphilic polymer, anamphiphilic diblock polymer, a protein, a nucleic acid, an epoxy, or apolysaccharide, and combinations thereof. The component can include apolymer. The polymer can include a polypeptide. The polypeptide caninclude at least a portion of an ELP elastin-like polypeptide (ELP).

In some embodiments, any component, or mixture of components can includean attached bioactive agent. The bioactive agent can include one or acombination of: a drug, a protein, a peptide, a peptide hormone, aligand, a cell-signaling ligand, or an RGD cell binding domain, to causerecruitment of the drug, protein, peptide, peptide hormone, ligand,cell-signaling ligand, or RGD cell binding domain to the coacervatedomain of a component. In an embodiment where a component is apolypeptide, the bioactive agent can be attached through an amino acidlinkage or through a chemical linkage through a reactive peptideresidue. The reactive peptide residue can include, for example, but isnot limited to lysine, cysteine, and aspartic acid. The polypeptideattached to the bioactive agent can include a protease cleavage site. Insome embodiments, the population of droplets are aqueous and thesolution includes one or a combination of a cell, a virus, or ananoparticle having a coating of at least one component to causerecruitment of the coated cell, virus, or nanoparticle to the coacervatedomain of the respective component within each of the droplets.

The phase separation point of any component, or mixture thereof, can bea phase separation temperature. For components or mixtures having aphase separation temperature, the stimulus can include heating. Thephase separation point can also include other phase separationcharacteristics, the characteristics relating to the stimulus applied toaffect a phase separation. For example, the stimulation can include:addition or removal of one or more of the components, evaporation of thedroplets or solution, controlled diffusion of one or more of thecomponents, electrostatic quenching of one or more of the components,inducing a reaction of one or more of the components, isomerization ofone or more of the components, crosslinking of one or more of thecomponent, or crystallization of one or more of the components, andcombinations thereof.

In some embodiments of the present disclosure, the solution includes atleast one surfactant for controlling a size of one or more coacervatedomains. The surfactant can include an amphiphilic diblock polymer. Forexample, but not limited thereto, a component can be a hydrophobic ELPpolymer and the amphiphilic diblock polymer can be an ELP diblockpolymer. By changing the ratio of a surfactant to a component, theresulting coacervate domain size can be manipulated. In someembodiments, a ratio of the hydrophobic ELP polymer to the amphiphilicELP diblock polymer can range from about 1:1 to about 50:1, which canresult in the size of an outermost coacervate domain ranging from about50 nm to about 20 μm.

In at least one embodiment, two or more components can have similarphase separation points. When stimulation induces a phase separationpoint of the two or more components, a blended alloy coacervate domaincan be formed. For example, but not limited thereto, where a first phaseseparation point of a first component and an additional phase separationpoint of an additional temperature are each similar phase separationtemperatures, and the stimulus includes heating, an alloy coacervatedomain can be formed. The coacervate domains of the various componentscan form a multilayered coacervate domain, a blended alloy coacervatedomain, or a combination thereof.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods described herein are presentlyrepresentative of preferred embodiments, are exemplary, and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the invention as defined by the scopeof the claims.

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The invention claimed is:
 1. A method for making nano- to microscaleliquid coacervate particles, the method comprising: stimulating apopulation of droplets including a solution of one or a mixture ofcomponents, wherein the stimulation induces a phase separation point ofa first component; and maintaining stimulation at the phase separationpoint to form a coacervate domain of the first component within each ofthe droplets, wherein liquid coacervate particles are formed.
 2. Themethod of claim 1, wherein the population of droplets are formed usingone or a combination of mechanical agitation, sonication, ormicrofluidics.
 3. The method of claim 1, wherein the population ofdroplets are aqueous droplets formed by sonication of the solution inoil or microfluidics of the solution in oil.
 4. The method of claim 1,wherein the liquid coacervate particles are reversibly formed bycessation of stimulation followed by re-stimulation and re-maintainingstimulation.
 5. The method of claim 1, wherein the first componentincludes a polymer.
 6. The method of claim 5, wherein the polymerincludes a polypeptide.
 7. The method of claim 6, wherein thepolypeptide includes at least a portion of an elastin-like polypeptide(ELP).
 8. The method of claim 1, wherein the one or a mixture ofcomponents includes: a polymer, a synthetic polymer, a hydrophilicpolymer, a hydrophobic polymer, an amphiphilic polymer, an amphiphilicdiblock polymer, a protein, a nucleic acid, an epoxy, or apolysaccharide, and combinations thereof.
 9. The method of claim 1,wherein the stimulating includes: addition or removal of one or more ofthe components, evaporation of the droplets, controlled diffusion of oneor more of the components, electrostatic quenching of one or more of thecomponents, inducing a reaction of one or more of the components,isomerization of one or more of the components, crosslinking of one ormore of the component, or crystallization of one or more of thecomponents, and combinations thereof.
 10. A method for coating asubstrate, the method comprising: stimulating a solution of one or amixture of components, wherein a substrate is immersed within thesolution, wherein the stimulation induces a phase separation point of afirst component; maintaining stimulation at the phase separation pointto form a degree of a coacervate domain of the first component on asurface of the substrate based on a wetting property of the substrate;and repeating the stimulating and maintaining for one or more additionalcomponents in the mixture to form a coacervate domain of the additionalcomponent.
 11. The method of claim 10, wherein the solution is aqueous.12. The method of claim 10, wherein the first component and theadditional component(s) include polymers.
 13. The method of claim 12,wherein the polymers include polypeptides.
 14. The method of claim 13,wherein the polypeptides include at least a portion of an elastin-likepolypeptide (ELP).
 15. The method of claim 10, wherein the firstcomponent and the additional component(s) have similar phase separationpoints and a blended alloy coacervate domain is formed on the surface ofthe substrate.
 16. The method of claim 10, wherein the substrateincludes one or more of a medical device, a stent, a vascular graft, acatheter, a biosensor, a drug reservoir or a cell culture substrate. 17.A nano- to microscale liquid coacervate particle composition produced bya process comprising: stimulating a population of droplets including asolution of one or a mixture of components, wherein the stimulationinduces a phase separation point of a first component; maintainingstimulation at the phase separation point to form a coacervate domain ofthe first component within each of the droplets, wherein liquidcoacervate particles are formed; and optionally repeating thestimulating and maintaining for the one or more additional components inthe mixture to form a coacervate domain of the additional componentwithin each of the droplets.
 18. The liquid coacervate particle of claim17, wherein the process further comprises stabilizing at least anoutermost coacervate domain within each of the droplets to form capsulestructures, wherein the outermost coacervate domain remains consolidatedupon cessation of stimulation at the phase separation point for theoutermost coacervate domain.
 19. The liquid coacervate particle of claim18, wherein stabilizing includes formation of cross-links by one or acombination of covalent coordination, ionic interaction, disulfidebonds, or hydrogen bonds.
 20. The liquid coacervate particle of claim17, wherein the first component and the additional components includepolymers.
 21. The liquid coacervate particle of claim 20, wherein thepolymers include polypeptides.
 22. The liquid coacervate particle ofclaim 21, wherein the polypeptides include at least a portion of anelastin-like polypeptide (ELP).
 23. The liquid coacervate particle ofclaim 17, wherein the first component and the additional component(s)have similar phase separation points and a blended alloy coacervatedomain is formed.
 24. The liquid coacervate particle of claim 17,wherein the population of droplets are aqueous droplets.